Ethanol- and PARP-Mediated Regulation of Ribosome-Associated Long Non-Coding RNA (lncRNA) in Pyramidal Neurons.

Although, by definition, long noncoding RNAs (lncRNAs) are not translated, they are sometimes associated with ribosomes. In fact, some estimates suggest the existence of more than 50 K lncRNA molecules that could encode for small peptides. We examined the effects of an ethanol and Poly-ADP Ribose Polymerase (PARP) inhibitor (ABT-888) on ribosome-bound lncRNAs. Mice were administered via intraperitoneal injection (i.p.) either normal saline (CTL) or ethanol (EtOH) twice a day for four consecutive days. On the fourth day, a sub-group of mice administered with ethanol also received ABT-888 (EtOH+ABT). Ribosome-bound lncRNAs in CaMKIIα-expressing pyramidal neurons were measured using the Translating Ribosome Affinity Purification (TRAP) technique. Our findings show that EtOH altered the attachment of 107 lncRNA transcripts, while EtOH+ABT altered 60 lncRNAs. Among these 60 lncRNAs, 49 were altered by both conditions, while EtOH+ABT uniquely altered the attachment of 11 lncRNA transcripts that EtOH alone did not affect. To validate these results, we selected eight lncRNAs (Mir124-2hg, 5430416N02Rik, Snhg17, Snhg12, Snhg1, Mir9-3hg, Gas5, and 1110038B12Rik) for qRT-PCR analysis. The current study demonstrates that ethanol-induced changes in lncRNA attachment to ribosomes can be mitigated by the addition of the PARP inhibitor ABT-888.

Effects of alcohol and PARP inhibition on RNA ribosomal engagement in cortical excitatory neurons.

We report on the effects of ethanol (EtOH) and Poly (ADP-ribose) polymerase (PARP) inhibition on RNA ribosomal engagement, as a proxy for protein translation, in prefrontal cortical (PFC) pyramidal neurons. We hypothesized that EtOH induces a shift in RNA ribosomal-engagement (RE) in PFC pyramidal neurons, and that many of these changes can be reversed using a PARP inhibitor. We utilized the translating ribosome affinity purification (TRAP) technique to isolate cell type-specific RNA. Transgenic mice with EGFP-tagged Rpl10a ribosomal protein expressed only in CaMKIIα-expressing pyramidal cells were administered EtOH or normal saline (CTL) i.p. twice a day, for four consecutive days. On the fourth day, a sub-group of mice that received EtOH in the previous three days received a combination of EtOH and the PARP inhibitor ABT-888 (EtOH + ABT-888). PFC tissue was processed to isolate both, CaMKIIα pyramidal cell-type specific ribosomal-engaged RNA (TRAP-RNA), as well as genomically expressed total-RNA from whole tissue, which were submitted for RNA-seq. We observed EtOH effects on RE transcripts in pyramidal cells and furthermore treatment with a PARP inhibitor "reversed" these effects. The PARP inhibitor ABT-888 reversed 82% of the EtOH-induced changes in RE (TRAP-RNA), and similarly 83% in the total-RNA transcripts. We identified Insulin Receptor Signaling as highly enriched in the ethanol-regulated and PARP-reverted RE pool and validated five participating genes from this pathway. To our knowledge, this is the first description of the effects of EtOH on excitatory neuron RE transcripts from total-RNA and provides insights into PARP-mediated regulation of EtOH effects.

Methylation and expression of glucocorticoid receptor exon-1 variants and FKBP5 in teenage suicide-completers.

A dysregulated hypothalamic-pituitary-adrenal (HPA) axis has repeatedly been demonstrated to play a fundamental role in psychiatric disorders and suicide, yet the mechanisms underlying this dysregulation are not clear. Decreased expression of the glucocorticoid receptor (GR) gene, which is also susceptible to epigenetic modulation, is a strong indicator of impaired HPA axis control. In the context of teenage suicide-completers, we have systematically analyzed the 5'UTR of the GR gene to determine the expression levels of all GR exon-1 transcript variants and their epigenetic state. We also measured the expression and the epigenetic state of the FK506-binding protein 51 (FKBP5/FKBP51), an important modulator of GR activity. Furthermore, steady-state DNA methylation levels depend upon the interplay between enzymes that promote DNA methylation and demethylation activities, thus we analyzed DNA methyltransferases (DNMTs), ten-eleven translocation enzymes (TETs), and growth arrest- and DNA-damage-inducible proteins (GADD45). Focusing on both the prefrontal cortex (PFC) and hippocampus, our results show decreased expression in specific GR exon-1 variants and a strong correlation of DNA methylation changes with gene expression in the PFC. FKBP5 expression is also increased in both areas suggesting a decreased GR sensitivity to cortisol binding. We also identified aberrant expression of DNA methylating and demethylating enzymes in both brain regions. These findings enhance our understanding of the complex transcriptional regulation of GR, providing evidence of epigenetically mediated reprogramming of the GR gene, which could lead to possible epigenetic influences that result in lasting modifications underlying an individual's overall HPA axis response and resilience to stress.

Differential H3K9me2 heterochromatin levels and concordant mRNA expression in postmortem brain tissue of individuals with schizophrenia, bipolar, and controls.

The existence of repressive and durable chromatin assemblies along gene promoters or networks, especially in the brain, is of theoretical and therapeutic relevance in a subset of individuals diagnosed with schizophrenia who experience a chronic, persistent, and treatment-resistant trajectory. We used chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) to generate an epigenomic map that includes differential sites occupied by di-methylated lysine 9 of histone 3 (H3K9me2), a repressive modification that is yet unexplored in human postmortem brain tissue. We have discovered over 150 significantly differential promoter sites in the postmortem prefrontal cortex tissue of individuals diagnosed with schizophrenia (n = 15) when compared to controls (n = 15). Potentially dysregulated gene categories include postsynaptic proteins, processing enzymes (for proproteins, lipids, and oxidative stress), cadherin family genes, the complement system, and peptide hormones. Ten genes with significantly increased or decreased H3K9me2 promoter occupation were selected through statistical analysis, function, or previous GWAS association, and Quantitative RT-PCR (qRT-PCR) was performed on an extended sample of postmortem brain tissue, adding an additional 17 controls, 7 individuals with schizophrenia, and 19 individuals with bipolar samples (n = 32 control, 22 schizophrenia, 19 bipolar). This approach revealed that mRNA expression levels correlated with chromatin modification levels in eight of 10 selected genes, and mRNA expression in the total sample could be predicted by the occupancy of H3K9me2. Utilization of this method and replication in a larger sample open a pathway to durable and restrictive epigenomic assemblies whose accumulation across the lifespan of individuals diagnosed with schizophrenia may explain treatment resistance, and advance therapeutic options.

Chemokines gene expression in the prefrontal cortex of depressed suicide victims and normal control subjects.

Abnormalities of neuroinflammation have been implicated in the pathogenesis of depression and suicide. This is primarily based on the observation that cytokines, which are major inflammatory molecules and play an important role in depression and suicide, are increased in both serum and in postmortem brain of depressed and suicidal subjects. Another class of immune mediators are chemokines which are primarily involved in chemotactic properties and trafficking of immune cells in the central nervous system (CNS). Chemokines also play an important role in CNS function. Whereas chemokines have been studied in the serum of depressed and suicidal patients, their role in brain of depressed or suicidal subjects is relatively unexplored. We studied the gene expression of several chemokines in the prefrontal cortex (PFC) obtained from depressed suicidal (DS) and normal control (NC) subjects. We determined the mRNA expression of several chemokines belonging to CXCL and CCL groups of chemokines using qPCR array technique and qPCR gene expression validation in 24 DS and 24 NC subjects. The postmortem brain samples were obtained from the Maryland Brain Collection. We found that the mRNA expression of chemokines CXCL1, CXCL2, CXCL3 and CCL2 was significantly decreased in the PFC of DS compared with NC subjects. No significant change was observed in CXCL5, CXCL6, CXCL10, CCL8 and CCL19 between DS and NC subjects. Since many of the chemokines are involved in mediating certain important CNS functions, such as neurotrophic effect, neurogenesis, anti-apoptotic growth factor release, modulation of synaptic transmission, brain development and neuronal loss, decreased levels of chemokines can reduce these functions which may be involved in the pathophysiology of depression.

Dysregulation of Protein Kinase C in Adult Depression and Suicide: Evidence From Postmortem Brain Studies.

BACKGROUND: Several lines of evidence suggest the abnormalities of protein kinase C (PKC) signaling system in mood disorders and suicide based primarily on the studies of PKC and its isozymes in the platelets and postmortem brain of depressed and suicidal subjects. In this study, we examined the role of PKC isozymes in depression and suicide. METHODS: We determined the protein and mRNA expression of various PKC isozymes in the prefrontal cortical region (Brodmann area 9) in 24 normal control subjects, 24 depressed suicide (DS) subjects, and 12 depressed nonsuicide (DNS) subjects. The levels of mRNA in the prefrontal cortex were determined by quantitative real-time reverse transcription PCR, and the protein expression was determined by western blotting. RESULTS: We observed a significant decrease in mRNA expression of PKCα, PKCßI, PKCδ, and PKCε and decreased protein expression in either the membrane or the cytosol fraction of PKC isozymes PKCα, PKCßI, PKCßII, and PKCδ in DS and DNS subjects compared with normal control subjects. CONCLUSIONS: The current study provides detailed evidence of specific dysregulation of certain PKC isozymes in the postmortem brain of DS and DNS subjects and further supports earlier evidence for the role of PKC in the platelets and brain of the adult and teenage depressed and suicidal population. This comprehensive study may lead to further knowledge of the involvement of PKC in the pathophysiology of depression and suicide.

Protein and mRNA expression of protein kinase C (PKC) in the postmortem brain of bipolar and schizophrenic subjects.

Abnormalities of protein kinase C (PKC) have been implicated in the pathophysiology of bipolar (BP) illness. This is primarily based on studies of PKC in platelets of BP patients. Whether such abnormalities of PKC activity and isoforms exist in the brain is unclear. We have therefore determined PKC activity, protein and mRNA expression of PKC isoforms in the prefrontal cortex (PFC), cingulate cortex (CING) and temporal cortex (TEMP) from BP (n = 19), schizophrenic (SZ) (n = 20) and normal control (NC) (n = 25) subjects. The brain samples were obtained from the Harvard Brain Bank, and the subjects were diagnosed according to DSM-IV criteria. Protein levels were determined using Western blot technique and mRNA levels were determined using real-time PCR (qPCR) method. We found that there was a significant decrease in the PKC activity in the cytosol and membrane fractions of PFC and TEMP obtained from BP subjects but not from SZ subjects. When we compared the expression of PKC isozymes, we found that the protein and mRNA expression of several isozymes was significantly decreased in the PFC (i.e., PKCα, PKCßI, PKCßII and PKCε) and TEMP (i.e., PKCα, PKCßI, PKCßII, PKCε and PKCγ) of BP subjects, but not in the CING. Overall, there was no difference in the mRNA or protein expression of PKC isozymes between SZ and NC subjects in any of the three brain areas we studied. Our results show that there is a region-specific decrease of certain PKC isozymes in the membrane and cytosol fractions of BP but not SZ subjects.

Increased protein and mRNA expression of corticotropin-releasing factor (CRF), decreased CRF receptors and CRF binding protein in specific postmortem brain areas of teenage suicide subjects.

Overactivity of hypothalamic-pituitary-adrenal (HPA) axis function has been implicated in depression and suicidal behavior. This is based on the observation of an abnormal dexamethasone (DEX) and DEX-adrenocorticotropic hormone (ACTH) test in patients with depression and suicidal behavior. Recently, some studies have also found abnormalities of glucocorticoid receptors (GR), mineralocorticoid receptors (MR), corticotropin releasing factor (CRF), CRF receptors (CRF-R) and CRF binding protein (CRF-BP) in depressed and suicidal patients. Some investigators have also observed increased levels of CRF in the cerebrospinal fluid (CSF) and altered levels of MR, GR and CRF in the postmortem brain of depressed and suicidal subjects. We have earlier reported decreased protein and mRNA expression of GR and GILZ, a chaperone protein, in the postmortem brain of teenage suicide subjects. We have further studied CRF and its receptors in different areas of the postmortem brain of suicide subjects, i.e., the prefrontal cortex (PFC), hippocampus (HIPPO), subiculum and amygdala (AMY) from teenage suicide subjects. The CRF and its receptors were determined in the PFC (Brodmann area 9), HIPPO, subiculum and different amygdaloid nuclei from 24 normal control subjects and 24 teenage suicide subjects. Protein expression of CRF, its receptors and CRF-BP was determined by immunolabeling using the Western blot technique and mRNA expression was determined by real-time PCR (qPCR) technique. We found that the mRNA levels of CRF were significantly increased in the PFC, in the central amygdaloid nucleus (CeAMY) and in the subiculum. mRNA levels of CRF-R1 and CRF-BP were significantly decreased in the PFC. We did not find any changes in the HIPPO of any of the CRF components we studied. When we compared the protein expression of CRF components we found that CRF was significantly increased and CRF-R1, CRF-R2 and CRF-BP significantly decreased in the PFC. On the other hand, there were no changes in the protein expression of CRF components in the HIPPO. Our results in the postmortem brain suggest that, as found by clinical studies in the CSF, there are significant alterations of CRF and its receptors in the postmortem brain of teenage suicide subjects. These alterations of CRF and its components were region-specific, as changes were not generally observed in the HIPPO.

Innate immunity in the postmortem brain of depressed and suicide subjects: Role of Toll-like receptors.

Abnormalities of Toll-like receptors (TLRs) have been implicated in the pathophysiology of depression and suicide. Interactions of TLRs with pathogen-associated molecular patterns (PAMP) and damage-associated molecular patterns (DAMP) initiate signaling through myeloid differentiation primary response-88 (MyD88) and produce cytokines through the activation of the transcription factor nuclear factor kappa beta (NF-kB). We have earlier shown an increase in the protein and mRNA expression of TLR3 and TLR4 in the prefrontal cortex (PFC) of depressed suicide (DS) subjects compared with normal control (NC) subjects. To examine if other TLRs are altered in postmortem brain, we have now determined the protein and mRNA expression of other TLRs (TLR1, TLR2, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10) in the PFC of DS, depressed non-suicide (DNS), non-depressed suicide (NDS) and NC subjects. We determined the protein expression by Western blot and mRNA expression levels by real-time PCR (qPCR) in the PFC of 24 NC, 24 DS, 12 DNS and 11 NDS subjects. Combined with our previous study of TLR3 and TLR4, we found that the protein expression of TLR2, TLR3, TLR4, TLR6 and TLR10, and mRNA expression of TLR2 and TLR3 was significantly increased in the DS group compared with NC group. This study demonstrated that certain specific TLRs are altered in DS subjects, and hence those TLRs may be appropriate targets for the development of therapeutic agents for the treatment of suicidal behavior.

Abnormal protein and mRNA expression of inflammatory cytokines in the prefrontal cortex of depressed individuals who died by suicide

Background: Depression and stress are major risk factors for suicidal behaviour, and some studies show abnormalities of proinflammatory cytokines in the serum and cerebrospinal fluid (CSF) of depressed and suicidal patients. However, it is not clear if similar abnormalities of cytokines are present in the brain of suicidal and depressed patients. Methods: We therefore determined the mRNA (using realtime polymerase chain reaction) and protein (using enzyme-linked immunosorbent assay and Western Blot) expression levels of interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)-α, lymphotoxin A, lymphotoxin B, IL-8, IL-10 and IL-13 in the prefrontal cortex (PFC) obtained from 24 depressed individuals who died by suicide and 24 nonpsychiatric controls. Results: We observed that the mRNA and protein levels of IL-1ß, IL-6, TNF-α, and lymphotoxin A were significantly increased, and levels of anti-inflammatory cytokine IL-10, and of IL-1 receptor antagonist (IL-1RA) were significantly decreased in the PFC of depressed individuals who died by suicide compared with controls. There were no significant differences in the protein and mRNA levels of IL-8 and IL-13 in the PFC. Limitations: The main limitation of this study is that some of the suicide group had been taking antidepressant medication at the time of death. Conclusion: Our results suggest that alterations of cytokines may be associated with the pathophysiology of depressed suicide and there may be an imbalance between pro- and anti-inflammatory cytokines in people who die by suicide. The causes of these increases in the brain of people who die by suicide, therefore, need to be investigated further.

Abnormal gene and protein expression of inflammatory cytokines in the postmortem brain of schizophrenia patients.

Immune function abnormalities have been implicated in the pathophysiology of schizophrenia (SZ). This is primarily based on the observation that the levels of proinflammatory cytokines are significantly increased in the serum of SZ patients compared with normal control (NC) subjects. However, it is not known if similar cytokines abnormalities are also present in the brain of SZ patients. To further examine the involvement of inflammatory cytokines in the brain of SZ patients, we determined the protein and mRNA levels of TNF-α, IL-1ß, IL-2, IL-6, IL-8, IL-10, IL-13, LTA and IL-1RA in the prefrontal cortex (PFC, Brodmann area 9) of SZ patients. We found that the protein and mRNA expression levels of the cytokines TNF-α and IL-6 are significantly increased and those of IL-10 are significantly decreased in the PFC of SZ patients. No difference in the protein and mRNA levels of IL-1ß, IL-13, and IL-1RA was observed between SZ patients and NC subjects. The protein expression levels of IL-8 were significantly decreased and those of LTA were significantly increased in SZ patients, but no significant difference in the mRNA levels of IL-8 and LTA was observed between SZ patients and NC subjects. The levels of IL-2 were undetectable or very low in the postmortem brain of either SZ or NC subjects. These results suggest abnormalities of specific pro- and anti-inflammatory cytokines in the postmortem brain of SZ patients. These observations may have important implications in understanding the role of inflammatory cytokines in the pathophysiology of SZ.

Expression of p21-activated kinases 1 and 3 is altered in the brain of subjects with depression.

The p21-activated kinases (PAKs) of group I are the main effectors for the small Rho GTPases, critically involved in neurodevelopment, plasticity and maturation of the nervous system. Moreover, the neuronal complexity controlled by PAK1/PAK3 signaling determines the postnatal brain size and synaptic properties. Stress induces alterations at the level of structural and functional synaptic plasticity accompanied by reductions in size and activity of the hippocampus and the prefrontal cortex (PFC). These abnormalities are likely to contribute to the pathology of depression and, in part, reflect impaired cytoskeleton remodeling pointing to the role of Rho GTPase signaling. Thus, the present study assessed the expression of the group I PAKs and their activators in the brain of depressed subjects. Using quantitative polymerase chain reaction (qPCR), mRNA levels and coexpression of the group I PAKs: PAK1, PAK2, and PAK3 as well as of their activators: RAC1, CDC42 and ARHGEF7 were examined in postmortem samples from the PFC (n=25) and the hippocampus (n=23) of subjects with depression and compared to control subjects (PFC n=24; hippocampus n=21). Results demonstrated that mRNA levels of PAK1 and PAK3, are significantly reduced in the brain of depressed subjects, with PAK1 being reduced in the PFC and PAK3 in the hippocampus. No differences were observed for the ubiquitously expressed PAK2. Following analysis of gene coexpression demonstrated disruption of coordinated gene expression in the brain of subjects with depression. Abnormalities in mRNA expression of PAK1 and PAK3 as well as their altered coexpression patterns were detected in the brain of subjects with depression.

Abnormal gene expression of proinflammatory cytokines and their membrane-bound receptors in the lymphocytes of depressed patients.

Abnormalities of protein levels of proinflammatory cytokines and their soluble receptors have been reported in plasma of depressed patients. In this study, we examined the role of cytokines and their membrane-bound receptors in major depressive disorder (MDD). We determined the protein and mRNA expression of proinflammatory cytokines, interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and mRNA expression of their membrane-bound receptors in the lymphocytes from 31 hospitalized MDD patients and 30 non-hospitalized normal control (NC) subjects. The subjects were diagnosed according to DSM-IV criteria. Protein levels of cytokines were determined by ELISA, and mRNA levels in lymphocytes were determined by the qPCR method. We found that the mean mRNA levels of the proinflammatory cytokines IL-1ß, IL-6, TNF-α, their receptors, TNFR1, TNFR2, IL-1R1 and the antagonist IL-1RA were significantly increased in the lymphocytes of MDD patients compared with NC. No significant differences in the lymphocyte mRNA levels of IL-1R2, IL-6R, and Gp130 were observed between MDD patients and NC. These studies suggest abnormal gene expression of these cytokines and their membrane-bound receptors in the lymphocytes of MDD patients, and that their mRNA expression levels in the lymphocytes could be a useful biomarker for depression.

The Expression of the Suicide-Associated Gene SKA2 Is Decreased in the Prefrontal Cortex of Suicide Victims but Not of Nonsuicidal Patients.

BACKGROUND: Recent study of genome-wide DNA methylation profiling in the postmortem brain of suicidal and nonsuicidal subjects found that gene expression of spindle and kinetochore associated complex subunit 2 (SKA2) is decreased in the postmortem brain of suicide victims compared with nonsuicidal, nonpsychiatric control subjects. METHODS: To determine if decreased SKA2 is specific to suicide and independent of diagnosis, we determined gene and protein expression of SKA2 in the prefrontal cortex obtained from suicide victims (n= 52), nonsuicidal psychiatric subjects (n= 27), and normal controls (n= 24). We determined gene expression by quantitative PCR technique and protein expression by Western blot. The postmortem brain samples were obtained from the Maryland Psychiatric Research Center. RESULTS: We found that protein and gene expression of SKA2 was significantly reduced in the prefrontal cortex of suicide victims compared with normal control subjects and nonsuicidal patients. We also found that SKA2 protein and gene expression in depressed suicide victims, schizophrenic suicide victims, and suicide victims with substance abuse and/or conduct disorders was significantly decreased compared with normal control subjects and also with nonsuicidal depressed or schizophrenic subjects. CONCLUSIONS: This study shows that decreased gene and protein expression of SKA2 observed in the prefrontal cortex of suicide victims is specific to suicide, which was not observed in the brain of nonsuicidal patients. It also indicates reduced SKA2 expression in suicide is independent of psychiatric diagnosis, since it is observed in all diagnostic groups studied. Therefore, SKA2 may be a potential biomarker for suicide.

Abnormal gene expression of proinflammatory cytokines and their receptors in the lymphocytes of patients with bipolar disorder.

OBJECTIVES: Abnormalities of protein levels of proinflammatory cytokines and their soluble receptors have been reported in plasma of patients with bipolar disorder (BP). In this study, we tested the hypothesis that the mRNA expression of membrane-bound receptors for proinflammatory cytokines will be altered in the lymphocytes of patients with BP. METHODS: We determined protein and mRNA expression of proinflammatory cytokines, and mRNA expression of their receptors in the lymphocytes from 29 drug-free, hospitalized patients with BP and 30 drug-free normal control subjects. The subjects were diagnosed according to DSM-IV criteria. Plasma protein levels of cytokines were determined by enzyme-linked immunosorbent assay (ELISA); mRNA levels in lymphocytes were determined by the quantitative polymerase chain reaction (qPCR) method. RESULTS: We found that mean mRNA levels of the proinflammatory cytokines interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α, and their receptors TNFR1, IL-1R1, and the antagonist IL-1RA were significantly higher in the lymphocytes of patients with BP compared with normal controls. CONCLUSIONS: This study suggests that the observed abnormalities of membrane-bound cytokine receptors may alter the functional response of cytokines in BP and that the mRNA levels of these receptors could be a potential biomarker.

Proinflammatory cytokines and their membrane-bound receptors are altered in the lymphocytes of schizophrenia patients.

Abnormalities of protein levels of proinflammatory cytokines and their soluble receptors have been reported in the plasma/serum of schizophrenia (SZ) patients. To examine if SZ is also associated with the abnormal gene expression of cytokines and their membrane-bound receptors, we studied mRNA expression of proinflammatory cytokines and their receptors in lymphocytes of SZ patients and normal control (NC) subjects. We determined the protein and mRNA expression of proinflammatory cytokines and mRNA expression of their receptors in lymphocytes from 30 SZ patients and 30 drug-free NC subjects. The subjects were diagnosed according to DSM-IV criteria. Protein levels of cytokines were determined by ELISA, and mRNA levels in lymphocytes were determined by the qPCR method. We found that the mRNA levels of IL-6, TNF-α, IL-1R1, TNFR1, and TNFR2, but not IL-1ß, IL-1R2, IL-1RA, IL-6R, or GP130 were significantly increased in lymphocytes of SZ patients compared with NC subjects. We also found that the protein expression of IL-6 and TNF-α, but not IL-1ß, was also significantly increased in SZ patients compared with NC subjects. These studies suggest that in addition to the reported abnormalities of proinflammatory cytokines and their soluble receptors in the plasma of SZ patients, an abnormal gene expression of these cytokines and their membrane-bound receptors may be involved in the pathogenesis of SZ.

Region-specific dysregulation of glycogen synthase kinase-3ß and ß-catenin in the postmortem brains of subjects with bipolar disorder and schizophrenia.

OBJECTIVES: There is both direct and indirect evidence suggesting abnormalities of glycogen synthase kinase (GSK)-3ß and ß-catenin, two important components of the Wingless-type (Wnt) signaling pathway, in the pathophysiology of bipolar illness and possibly schizophrenia (SZ). In order to further clarify the role of the Wnt signaling pathway in the pathophysiology of bipolar disorder (BP) and SZ, we studied GSK-3ß and ß-catenin in the postmortem brains of subjects with these disorders. METHODS: We determined the protein expression of GSK-3ß, phosphorylated form at serine 9 position (pGSK-3-ser-9), and ß-catenin using the western blot technique, and mRNA using the quantitative polymerase chain reaction (qPCR) method, in the dorsolateral prefrontal cortex (DLPFC), cingulate gyrus (CG), and temporal cortex (TEMP) obtained from 19 subjects with BP, 20 subjects with SZ, and 20 normal control (NC) subjects. RESULTS: We found that the protein expression of GSK-3ß, pGSK-3ß-ser-9, and ß-catenin was significantly decreased in the DLPFC and TEMP, but not in the CG, of subjects with BP compared with NC subjects. The mRNA expression of GSK-3ß and ß-catenin was significantly decreased in the DLPFC and TEMP, but not in the CG, of subjects with BP compared with NC subjects. There were no significant differences in the protein or mRNA expression of GSK-3ß, pGSK-3ß-ser-9, or ß-catenin between subjects with SZ and NC subjects in any of the brain areas studied. CONCLUSIONS: These studies show region-specific abnormalities of both protein and mRNA expression of GSK-3ß and ß-catenin in postmortem brains of subjects with BP but not subjects with SZ. Thus, abnormalities of the Wnt signaling pathway may be associated with the pathophysiology of bipolar illness.

Toll-like receptors in the depressed and suicide brain.

Abnormalities of the immune function in depression and suicide are based in part on the observation of increased levels of proinflammatory cytokines in the serum and postmortem brain of depressed and suicidal patients. We have examined if abnormalities of the innate immune receptors, known as Toll-like receptors (TLRs), in the brain are associated with depression and suicide, since the activation of these receptors results in production of cytokines. Of all the TLRs shown to be present in humans, TLR3 and TLR4 appear to be unique and important in brain function. We have determined the protein (by ELISA method) and mRNA expression (using qPCR) of TLR3 and TLR4 in the postmortem brain (dorsolateral prefrontal cortex [DLPFC]) of 22 depressed suicide victims, 11 non-depressed suicide victims, 12 depressed non-suicide subjects and 20 normal control subjects. We found that the mRNA expression of TLR3 and TLR4 was significantly increased in DLPFC of depressed suicide victims and in depressed non-suicide subjects, compared with controls. However, the protein expression of TLR3 and TLR4 was significantly increased in depressed suicide victims, but not in depressed non-suicide subjects compared with controls. The observed abnormalities of proinflammatory cytokines in the brain of suicide victims may be related to an abnormality of TLR3 and TLR4 over-expression. To our knowledge, this is the first study of TLRs in the brain of psychiatric subjects.

Alteration of cyclic-AMP response element binding protein in the postmortem brain of subjects with bipolar disorder and schizophrenia.

BACKGROUND: Abnormalities of cyclic-AMP (cAMP) response element binding protein (CREB) function has been suggested in bipolar (BP) illness and schizophrenia (SZ), based on both indirect and direct evidence. To further elucidate the role of CREB in these disorders, we studied CREB expression and function in two brain areas implicated in these disorders, i.e., dorsolateral prefrontal cortex (DLPFC) and cingulate gyrus (CG). METHODS: We determined CREB protein expression using Western blot technique, CRE-DNA binding using gel shift assay, and mRNA expression using real-time RT-polymerase chain reaction (qPCR) in DLPFC and CG of the postmortem brain of BP (n=19), SZ (n=20), and normal control (NC, n=20) subjects. RESULTS: We observed that CREB protein and mRNA expression and CRE-DNA binding activity were significantly decreased in the nuclear fraction of DLPFC and CG obtained from BP subjects compared with NC subjects. However, the protein and mRNA expression and CRE-DNA binding in SZ subjects was significantly decreased in CG, but not in DLPFC, compared with NC. CONCLUSION: These studies thus indicate region-specific abnormalities of CREB expression and function in both BP and SZ. They suggest that abnormalities of CREB in CG may be associated with both BP and SZ, but its abnormality in DLPFC is specific to BP illness.

Region-specific alterations in glucocorticoid receptor expression in the postmortem brain of teenage suicide victims.

INTRODUCTION: Abnormal function of the hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the pathophysiology of depression and suicide. The purpose of this study was to test the hypothesis that the reported dysregulation of the HPA axis in suicide may be related to a disturbed feedback inhibition caused by decreased corticoid receptors in the brain. We therefore determined the protein and gene expression of glucocorticoid (GR) and mineralocorticoid receptors (MR) in the postmortem brain of teenage suicide victims and matched normal controls. METHODS: Protein and mRNA expression of GR (GR-α and GR-ß) and MR and the mRNA expression of glucocorticoid-induced leucine zipper (GILZ), a target gene for GR were determined by immunolabeling using Western blot technique and the real-time RT-polymerase chain reaction (qPCR) technique in the prefrontal cortex (PFC), hippocampus, subiculum, and amygdala obtained from 24 teenage suicide victims and 24 teenage control subjects. RESULTS: We observed that protein and gene expression of GR-α was significantly decreased in the PFC and amygdala, but not in the hippocampus or subiculum, of teenage suicide victims compared with normal control subjects. Also, the mRNA levels of GR inducible target gene GILZ was significantly decreased in PFC and amygdaloid nuclei but not in hippocampus compared with controls. In contrast, no significant differences were observed in protein or gene expression of MR in any of the areas studied between teenage suicide victims and normal control subjects. There was no difference in the expression of GR-ß in the PFC between suicide victims and normal controls. CONCLUSIONS: These results suggested that the observed dysregulation of the HPA axis in suicide may be related to a decreased expression of GR-α and GR inducible genes in the PFC and amygdala of teenage suicide victims. The reason why GR receptors are not dysregulated in the hippocampus or subiculum, presumably two sites of stress action, are not clear at this time.